Immortality is of primary importance to human breast carcinogenesis as a prerequisite for accumulation of neoplastic changes in breast cells. In this proposal, cultured human mammary epithelial cells (HMEC) will be used to study changes contributing to immortalization. Fully immortal HMEC and breast tumor-derived cell lines often significantly overexpress telomere-binding protein, TRF2. High levels of endogenous TRF2 may be a consequence of the cells attempting to protect their critically short telomeres from being recognized as damaged DNA during the early stages of immortalization. Up-regulated TRF2 may then facilitate proliferation by telomere protection, telomere length regulation, and cell cycle checkpoint inhibition in the face of DNA damage. The objectives of this proposal are: 1) to determine whether up-regulation of TRF2 in fully immortal HMEC correlates with changes in protein-protein interactions, increased TRF2 stability, or changes in proteolytic processing; 2) to investigate whether up-regulated TRF2 is a consequence of critically short telomeres or derepression of telomerase; and 3) to elucidate whether TRF2 overexpression leads to perturbation of the cell cycle checkpoints by sequestering DNA damage sensors ATM and ATR. Results obtained in this proposal will elucidate processes occurring during early breast carcinogenesis. These studies also hold potential to identify TRF2 overexpression as a new diagnostic marker and a possible target for therapeutic intervention in the early stages of breast carcinogenesis.